During the past grant period we used amino acid sequences from the cholinergic differentiation factor (CDF) to clone the rat cDNA for the cytokine also known as leukemia inhibitor factor (LIF). Using CDF/LIF and other recombinant proteins, and a novel cell culture assay, our work contributed to the recognition of the neuropoietic cytokine family. We continue to look for novel members of this family and have begun to explore the in vivo role of LIF in development, in the response to neural injury, and in the synaptic plasticity involved in LTP. LIF: (1) Our study of LIF-mutant mice reveals atrophic changes in the hippocampus and the visual cortex. These observations will be extended to the examination of brains from developing animals and the use of many other phenotypic markers. We have also obtained embryonic brains of LIFR-mice, and brains of rats that were injected with LIF-blocking antibodies at birth; the rats display behavioral abnormalities. In complementary experiments, we will inject LIF-secreting fibroblasts into the brains of normal and LIF-mice of various ages. (2) To determine if LIF acts directly on CNS neurons, it will be tested on cultured hippocampal and cortical neurons for effects on survival and gene expression. (3) We find that LIF expression is strongly induced in glial cells upon nerve injury, and that the neuropeptide induction that normally follows nerve section is much reduced in LIF-mice. We will now examine whether neuronal survival and the rate of nerve regeneration are altered following nerve section in LIF-mice. We will also investigate whether IL-1 mediates the LIF induction following injury in vivo. (4) We have begun to explore the role of LIF in neurogenic inflammation. We find that LIF is induced in the skin by intradermal CFA, a paradigm that leads to neuropeptide induction in sensory neurons. We will test whether this neuropeptide induction also occurs in LIF-mice subjected to CFA. (5) Since LIF and its receptor mRNAs are found in the hippocampus, and LIF mRNA is elevated by high levels of activity, we will ask if this protein has a role in LTP. The effects of LIF and its blocking antibody on synaptic physiology in hippocampal slices will be monitored, and LIF mRNA will be assayed following LTP. Preliminary results indicate that LIF suppresses the induction of LTP in slices. New Cytokines: (1) We are searching for homologues of GPA, CNTF and LIF. Bands of the predicted sizes have been obtained using the PCR for all three cytokines in chick, mammal and fish, and these are being characterized. Northern analysis, cloning full cDNAs and protein expression are planned. (2) Since another neuropoietic cytokine, OSM, is only available from human, very little is known about its potential role in the nervous system. We are attempting to clone the mouse OSM gene using nested PCR primers, and would like to examine OSM expression in the normal and injured PNS and CNS.